The combined use of TLR and CD40 agonists together with tumor antigen is a novel vaccine platform that elicits robust immunity resulting in the effective eradication of pre-existing cancer. The synergy that is manifested by the combined administration of both CD40 and TLR agonists (TLR*) is represented by an greatly enhanced frequency of atumor effector T cells. Maximizing this approach to provide improved therapeutic protection in cancer, understanding the mechanisms underlying the generation of tumor-specific effector T cells and the testing of this vaccine platform approach in multiple, clinically-relevant models of melanoma will be sought (Specific Aim#1). While understanding how an effective cancer vaccine can elicit potent atumor effector T cells is important, it is equally important to resolve how it limits the emergence of regulatory T cells (Treg). Ample evidence establishes that natural (n)Treg and adaptive (a)Treg can dramatically influence the immune responses to tumor. Our data shows that only when both aCD40 and TLR* signals are provided can one limit the emergence of tumor-specific aTreg, and this may be key to the development of protective atumor immunity. Cytokines, cells and molecules that control the differentiation of tumor-specific aTreg will be identified and the mechanisms of how aTreg development influences effective atumor immunity will be studied (Specific Aim #2). Together, Specific Aims #1 and #2 should provide a mechanistic understanding of how to maximize immune effector mechanisms and limit immune regulation to tumor-specific antigens using the aCD407TLR* platform. Enhancing immune effector mechanisms and dampening immune regulation may be controlled by a new cellular circuitry involving Treg, IL-9 and mast cells. Our studies show that mast cells are essential intermediaries in CD4+CD25+Foxp3+ Treg-dependent peripheral tolerance. Contrary to the pro-inflammatory role played by mast cells, we have reported that mast cells are critical in mediating peripheral suppression. High levels of IL-9 (mast cell growth factor) produced by activated Treg is important in mast cell recruitment and activation in tolerant tissue. IL-9 represents the functional link through which activated Treg recruit and activate mast cells to mediate regionalized immune suppression. We present data that Treg, mast cells and IL-9 also play a role in down- regulating the immune response to tumors. We hypothesize that effective immune intervention will impact on the Treg -IL-9-mast cell axis leading to protective tumor immunity (Specific Aim #3). Developing an understanding of how to implement and deliver this novel adjuvant platform into the clinic for use in the creation of an effective atumor vaccine will provide new avenues for the inducing immunity to cancers.